Antimicrobial peptides

ABSTRACT

The invention provides a peptide comprising: a core amino acid sequence, which is identical or similar to the amino acid sequence of a member of the Cecropin family. The invention further provides a nucleic acid sequence encoding the peptide and a vector comprising said nucleic acid. The invention further provides a pharmaceutical composition comprising said peptide or said nucleic acid. The invention further provides methods of treating an infection, overcoming inherent or acquired resistance of a microorganism to an antibiotic agent or disinfecting a wound, the methods comprises administering the peptide to a subject in need thereof.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/115,161 filed on Jul. 28, 2016, which claims the benefit of NationalPhase of PCT Patent Application No. PCT/IL2016/050187 havingInternational filing date of Feb. 17, 2016, and of U.S. ProvisionalApplication No. 62/119,186 filed on Feb. 22, 2015. The contents of theabove applications are all incorporated by reference as if fully setforth herein in their entirety.

FIELD OF INVENTION

The invention encompasses antimicrobial peptides for therapeutic uses.These peptides are based on the Cecropin family which serves as potentantibacterial agents in insects.

BACKGROUND OF THE INVENTION

Antibiotics are chemical substances having the capacity, in a dilutesolution, to kill or inhibit growth of microorganisms. Antibiotics thatare sufficiently nontoxic to the host are used as chemotherapeuticagents to treat infectious diseases of humans, animals, and plants. Theterm was originally restricted to substances produced by microorganisms,but has been extended to include synthetic and semi-synthetic compoundsof similar chemical activity.

Extensive and widespread use of antimicrobial drugs led to the emergenceof resistant strains of microorganisms. These microorganisms are nolonger susceptible to currently available antimicrobial drugs. In orderto lower or prevent lethal infectious diseases and maintain publichealth, new antimicrobial agents are required. This forces researchersto pursue novel antibiotics, not yet resistant by bacteria.Antimicrobial peptides (AMPs) are part of the armament that insects havedeveloped to fight off pathogens. Although usually cationic, the primarystructures of insect AMPs vary markedly. Members of the most frequentAMP families adopt an α-helical conformation in membrane-mimeticenvironments (Bulet P. et al., Protein and Peptide Letters, 2005, 12,3-11).

Insects produce antibacterial peptides, which are secreted to theirhemolymph, as an innate defense against pathogenic infections (Boman, H.G. et al., Annu. Rev. Microbial., 1987, 41, 103-126). Some insectspecies are capable of producing 10-15 different antibiotic peptides(Hoffman, J. A., et al., FEBS Let., 1993, 325, 663-664). Each peptidehas a complete different range of antibacterial action (Bulet, P.Medicine Sciences 1999. 15, 23-29).

Cecropins were first isolated from the hemolymph of Hyalophora cecropia.Cecropins are small cationic peptides consisting 29-42 amino acidresidues, found in the Diptera order (genus Drosophila, Sarcophaga) andLepidoptera order (genus Hyalophora, Manduca, Bombyx, Antheraea). Itshould be mentioned that a Cecropin was isolated from porcine intestine(Boman, H. G., et al. Eur. J. Biochem. 1991. 201, 23-31; Morishima, I.,et al. Biochem. Physiol. 1990. 95B, 551-554; Steiner, H., et al. Nature1981. 292, 246-248; Sun, D., et al. Biochem. Biophys. Res. Commun. 1998.249(2), 410-415; Bulet, P. et al Immunological Reviews. 2004. 198,169-184). The known sequences for the major Cecropins show that theN-terminal parts are strongly basic while the C-terminal regions areneutral and contain long hydrophobic stretches. In all cases theCecropins have an amidated C-terminal residue (Boman, H. G. et al.,Annu. Rev. Microbial., 1987, 41, 103-126). Cecropins secondary structureforms two amphiphatic α-helixes which are able to penetrate thebacterial membrane. This ability is followed by membrane loss of ionicgradient balance leading to bacterial death (Christensen, B. C., et al.Proc. Natl. Acad. Sci. USA. 1988 83:1670-1674; Lockey, T. D., et al.Eur. J. Biochem. 1996. 236, 263-271; Marassi, F. M., et al. Biophys. J.1999. 77, 3152-3155; Wang, W., et al. J. Biol. Chem. 1998. 273, (42)27438-27448).

Cecropins are very similar molecules as half the amino acidsubstitutions are strictly conservative. Theoretical predictions andcircular dichroism spectra indicate that these peptides can form nearlyperfect amphipathic α-helices with charged groups on one longitudinalside and hydrophobic side residues on the opposite side. Proteins withamphipathic helices are often associated with membranes, and thissecondary structure may be of importance for the membrane-disruptingactivity of the Cecropins (Boman, H. G. et al., Annu. Rev. Microbial.,1987, 41, 103-126).

The structure of different sequences of peptides of the Cecropin familyshows that they represent similar types of molecules. In addition tostrongly basic N-terminal region and a long hydrophobic stretch in theC-terminal half, there are other typical conserved features such as:tryptophan at position 2, the single and double lysines at positions 5,8 and 9 and arginine at position 12. It can be concluded that there musthave been strong selection pressures that have conserved certainCecropin sequences in different types of insects throughout evolution(Boman, H. G., et al. Eur. J. Biochem. 1991. 201, 23-31).

Membrane-active peptides exhibit channel-like conductivities acrossplanar lipid bilayer systems as well as bilayer disruption. Thesebilayer openings deprive the affected organisms of their transmembraneelectrochemical gradients, resulting in increased water flow concomitantwith cell swelling, osmolysis and cell death. Antimicrobial peptides ofparticular interest for pharmacological applications are those whichmanifest antibacterial activity, but under the same conditions, do notshow hemolytic or cytotoxic effect against healthy vertebrate cells (B.Bechinger. et al. J. Membrane Biol. 1997.156, 197-211). Mostantibacterial peptides have to be positively charged in order to bind tobacterial surfaces, which normally are negatively charged. Cecropinsshow strong antibiotic activity against a variety of Gram-negative andGram-positive bacteria without lysing mammalian cell lines or yeast(Agerberth, B. et al. Eus. J. Biochem. 1993. 216, 623-629).

The cell killing activity of Cecropins is not mediated through specific,chiral receptor interactions. The cell lytic activity of these peptidescorrelates with their ability to form α-helical secondary structures inmembrane environments as well as with their binding affinity toliposomes (B. Bechinger. et al. J. Membrane Biol. 1997.156, 197-211).Toxicity studies on a variety of cell types have shown that, althoughplant protoplasts are more sensitive to Cecropins than are animal cells,plant cells are one to two orders of magnitude less sensitive to thesepeptides than their bacterial pathogens (Jaynes, J. M., et al. PeptideRes. 1989. 2, 157-160; Nordeen, R. D., et al. Plant Sci. 1992. 82,101-107).

A strong example for Cecropin advantage as antimicrobial agents can befound in Cecropin A. Cecropin A, a 37-residue peptide, is composedentirely of ordinary

L-amino acids (Steiner H., et al. Nature. 1981, 292:246-248). Cecropin Asecondary structure is composed of two amphiphatic α-helixes with anidentical length of bacterial plasma membrane. The primary target ofthis toxin is assumed to be the microbial membrane, and itsantimicrobial effect is probably due to ionophore activity. WhenEscherichia coli bacteria were treated with Cecropin A, K⁺ ions insideof the cells leaked out rapidly and the ATP pool of the cells rapidlydecreased. These results suggested that the bactericidal effect ofCecropin A was due to its ionophore activity, and that it blocked thegeneration of ATP by inhibiting formation of the proton gradientessential for oxidative phosphorylation (Natori, S. Nippon Rinsho. 1995.53, 1297-1304; Okada, M., et al. Biochem. J. 1985.229, 453-458,Silvestro L. et al. Antimicrob Agents Chemother. 2000 March; 44(3):602-607).

It should be noted that Cecropins inhibited the growth of harmfulbacteria in the human intestine without affecting the growth ofbeneficial bacteria which are abundant in the intestines of healthypeople (Mitsuhara, I., et al. Biotechnology Letters. 2001. 23, 569-573).

The use of peptides as antibiotics is not obvious due to theirsensitivity to protease activity (Andrew, D., et al. Biopolymers.1998.47, 415-433). Most Cecropins are rich with Lysine and Arginineresidues, which commonly comprise part of target sequences for abundantproteases such as trypsin, Inhibitor A and Proteinase K (GunnelDALHAMMAR et al. Eur. J. Biochem. 139, 247-252 (1984, Bland J M et al.Journal of agricultural and food chemistry 1998 v. 46 no. 12 pp.5324-5327). Previous research has shown that Cecropins are rapidlydegraded in the intracellular fluid of plants (Owens, L. D., et al. Mol.Plant Microbe Interact. 1997. 10, 525-528). Several experiments tryingto express Cecropins in plants have failed probably due to sensitivityto proteolytic activity (Allefs, S. J. H. M., et al. Am. Potato J. 1995.72, 437-445; Florack, D., et al. Transgenic Res. 1995. 4, 132-141;Hightower, R., et al. Plant Cell Rep. 1994. 13, 295-299).

The engineering of stable proteins is of great technological andeconomic importance, since the limited stability of proteins oftenseverely restricts their medical and industrial application. It istherefore an object of the invention to provide novel stablepeptide-based antibiotics, such as AMCP's.

SUMMARY OF THE INVENTION

The present invention provides genetically engineered or synthesizeddegradation-resistant, peptides. In some embodiments, the peptidescomprise at least one cysteine residue at their carboxy- andamino-terminus. In some embodiments of the invention, under oxidativeenvironment, e.g. as in various infections, the cysteines in thecarboxy- and amino-terminus of the peptides of the invention, arecovalently bonded, thus creating in an embodiment of the invention acyclic form of the peptides, wherein said cyclic peptides representhigher stability while maintaining its original biological activity.

The present invention relates to a peptide comprising a core amino acidsequence, which is identical or similar to the amino acid sequence of amember of the Cecropin family, wherein the core amino acid sequence isextended at the N-terminus by an N-terminal group and/or extended at theC-terminus by a C-terminal group; and wherein the N-terminal groupand/or the C-terminal group are identical or different and are capableof forming a covalent bond so as to form a cyclic peptide or ahomomultimer assembly via intermolecular covalent linkage.

In some embodiments of the invention, the member of the Cecropin familybelongs to the group of AMP CM_(IV), Cecropin A, Cecropin B, CecropinB2, Cecropin D, Cecropin IA, and Cecropin P1.

In some embodiments of the invention, the cyclic peptide has a topology,wherein the topology is head-to-tail, side-chain-to-side-chain,head-to-side-chain or side-chain-to-tail or backbone-to-backbone orside-chain-to-backbone or head-to-backbone or tail-to-backbone.

In some embodiments of the invention, the covalent linkage is formedunder oxidative and/or acidic physiological conditions.

In some embodiments of the invention, the amino acid sequence of themember of the Cecropin family comprises 17-144 amino acids.

In some embodiments of the invention, the core amino acid sequence of amember of the Cecropin family is as set forth in SEQ ID Nos. 12-22.

In some embodiments of the invention, the core amino acid sequence hasat least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to theamino acid sequences set forth in SEQ ID Nos. 12-22.

In some embodiments of the invention, the core amino acid sequencecomprising substitution, conservative amino acid substitutions,conservatively modified sequence variants, deletion, and/or insertion atone or more position.

In some embodiments of the invention, the C-terminus group and/or the Nterminus group comprises one or more of cysteine, cysteine derivative,an amino acid sequence, which contains cysteine or a group comprising athiol moiety or any combination thereof.

In some embodiments of the invention, the C-terminus group and/or the Nterminus group are each selected from the group consisting of cysteine,cysteine derivative, an amino acid sequence which contains cysteine orany other group comprising a thiol moiety.

In some embodiments of the invention, the N-terminus group comprises theamino acid sequence methionine-cysteine, methionine-cysteine derivative,methionine derivative-cysteine or methionine derivative-cysteinederivative and the C-terminus group is cysteine or a cysteinederivative.

In some embodiments of the invention, the C-terminus group comprises theamino acid sequence methionine-cysteine, methionine-cysteine derivative,methionine derivative-cysteine or methionine derivative-cysteinederivative and the N-terminus group is cysteine or a cysteine derivative

In some embodiments of the invention, the covalent linkage is adisulfide bond.

In some embodiments of the invention, the covalent linkage is an amide,lactam or peptide bond.

In some embodiments of the invention, the N-terminus group and theC-terminus group are covalently bound so as to form a cyclic peptide.

In some embodiments of the invention, the peptide is self-assembledwithin a physiological membrane such that the intermolecular covalentlinkage is formed between the N-terminus group of the peptide to theN-terminus or a C-terminus group of an additional identical peptide orwherein the intermolecular covalent linkage is formed between theC-terminus group of the peptide to the N-terminus or a C-terminus groupsof an additional identical peptide.

In some embodiments of the invention, the peptide is as set forth in anyone of SEQ ID Nos. 1-11.

In some embodiments of the invention, the peptide has an amino acidsequence which is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequenceidentity to the amino acid sequences set forth in SEQ ID Nos. 1-11.

In some embodiments of the invention, the peptide is as set forth in SEQID NO: 6.

In some embodiments of the invention, the peptide has an amino acidsequence which is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 6.

The present invention relates to a nucleic acid sequence encoding anyone of the above referenced peptides.

The present invention relates to a vector comprising the abovereferenced nucleic acid.

The present invention relates to a pharmaceutical composition comprisingany one of the above referenced peptides or the above referenced nucleicacid.

The present invention relates to a method of treating an infection, themethod comprising administering and one of the above referenced peptidesor the above referenced pharmaceutical composition to a subject in needthereof.

The present invention relates to a use of any one of the abovereferenced peptides or the above referenced pharmaceutical compositionin the preparation of a medicament for treating an infection in asubject.

In some embodiments of the invention, the infection is bacterial, viral-and/or fungal infection.

In some embodiments of the invention, the pharmaceutical composition isin a form of a liquid, cream, gel, paste, powder, emulsion, an ointment,a liniment, a lotion, a transdermal system, an injection fluid, asuspension, a patch film patch or spray.

In some embodiments of the invention, the pharmaceutical composition isin the form of capsule or a tablet.

In some embodiments of the invention, the composition or the peptide isadministered in conjunction with one or more additionalanti-inflammatory active agent.

The present invention relates to a method of overcoming inherent oracquired resistance of a microorganism to an antibiotic agent,comprising: contacting the microorganism to any one of the abovereferenced peptides or the above referenced pharmaceutical composition.

In some embodiments of the invention, the microorganism is Escherichiacoli, Klebsiella pneumoniaea, Pseudomonas aeruginosa, Salmonellaserotype Typhi, Acinetobacter baumannii, a member of Enterobacteriaceaespp., Pseudomonas spp. Salmonella spp., or Acinetobacter spp., or anycombination thereof.

The present invention relates to a method of disinfecting a woundcomprising contacting the wound with any one of the above referencedpeptides or the above referenced pharmaceutical composition.

In some embodiments of the invention, the wound is a blister wound, asoft tissue wound, a cutaneous abscess, a surgical wound, a suturedlaceration, a contaminated laceration, a burn wound, a decubitus ulcer,a stasis ulcer, a leg ulcer, a foot ulcer, a venous ulcer, a diabeticulcer, an ischemic ulcer, a pressure ulcer, an oral infection, aperiodontal disease, a partial thickness burn, or a full thickness burn.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts a Coommassie Blue (Brilliant Blue R, Sigma ChemicalCompany, USA) stained gel detecting the following: Lanes 1-3: NativeCecropin-A (CecA) subjected to Proteinase-K (ProtK) proteolysis inincreasing concentrations: 0/5/20 ng/30 μl as mentioned. Lanes 4-6: OMN6subjected to ProtK proteolysis in increasing concentrations: 0/5/20ng/30 μl as mentioned. Lanes 7-9: Bovine Serum Albumin (BSA) subjectedto ProtK proteolysis in increasing concentrations: 0/5/20 ng/30 μl asmentioned. Proteolysis of CecA and BSA resulted in the disappearing ofthe band from the gel. Proteolytic activity of ProtK did not affect OMN6and all the bands were present.

FIG. 1B depicts Coommassie blue stained gel detecting the stability ofpeptides OMN2, OMN7 and OMN11 against ProtK activity. The stability ofeach peptide with or without incubation with ProtK was presented (rightlane and left lane, respectively). Clearly, peptides OMN2, OMN7 andOMN11 were not degraded by the depredating activity of ProtK. The bandsare of the same intensity either for the ProtK treated peptide as forthe non-treated peptide, namely, ProtK had no depredating effect onthese peptides.

FIG. 2A: Right graph demonstrates E. coli bacteria growth monitored over17.5 hours via absorbance at 600 nm. Bacteria growth or inhibition ofgrowth is presented as OD600 nm values over time. As the bacteria grow,OD values increase. OMN6 antimicrobial effect is demonstrated by thedecreased OD values that represent bacterial death in the presence ofOMN6. Left graph same experiment as described above was conducted withE. coli bacteria and the native peptide Cecropin A (CecA). As is clearlyseen after 10 hours CecA loses its antimicrobial activity and becomesineffective. At that point, the bacteria overcome the growth inhibitionand begin to grow and flourish. At the end of the experiment, bacteriatreated with CecA reach the density and growth levels similar to that ofthe CTRL group (treated with DDW). These results strongly point to thefact that OMN6 is a stronger antimicrobial agent than CecA.

FIG. 2B depicts bacterial survival as percent survival of the controlgroup (%/CTRL) in presence of CecA or one of the peptides of theinvention, OMN6. Left Bars demonstrate bacterial survival in thepresence of 12.5 μM of CecA pre-treated with 20 ng of ProtK. Right Barsdemonstrate bacterial survival in the presence of 12.5 μM of OMN6pre-treated with 20 ng of ProtK. CecA is degraded by ProtK therefore thebacteria survival is more than 70% of the CTRL group. OMN6 is stable andactive, and accordingly the bacteria growth is inhibited to less than10% of the CTRL group.

FIG. 3A: is a photograph showing Green Fluorescent Protein (GFP) leakingfrom lysed bacteria cells. FIG. 3A shows bacteria treated with DDW as acontrol (CTRL). As can be seen the bacteria are alive, well defined,intact and GFP fluorescence is detected only inside the bacteria.

FIG. 3B shows bacteria treated with OMN6. The bacteria are dead, andhave undergone extensive lysis, which allowed the GFP fluorescence to bedetected outside of bacteria cells, in the surrounding media.

FIG. 4A Left Panel is a photograph showing the leaking of GFP from lysedbacteria to the surrounding media, after centrifugation with or withouta treatment with OMN6. Bacteria in the left tube, receiving DDW shamtreatment are alive, intact and all GFP fluorescence is limited tobacteria cells in pellet. Bacteria in right tube, receiving treatmentwith OMN6, have undergone extensive lysis and GFP fluorescence isclearly visible outside of dead bacterial cells and in the surroundingmedia.

FIG. 4B demonstrates the graphical quantitation of the FluorescenceUnits (FU) in each group.

FIGS. 5A-5D: present the survival of HEK293 cells quantified viaMethylene-Blue assay and presented as percent survival of CTRL (%/CTRL).A 24 hours treatment with increasing concentration of OMN2 (FIG. 5A),OMN6 (FIG. 5B), OMN7 (FIG. 5D) and OMN11 (FIG. 5C) does not lead to celldeath or alteration of the survival fraction.

FIG. 6A: E. coli bacteria growth monitored over 17.5 hours viaabsorbance at 600 nm. Bacteria growth or inhibition of growth ispresented as OD600 nm values over time. As the bacteria grow, OD valuesincrease. A dose-response of OMN6 antimicrobial effect is demonstratedby the decreased OD values that represent bacterial death in correlationwith increasing OMN6 concentration in μM.

FIG. 6B: The same experimental system as described for FIG. 6A wasapplied, with the addition of 10% Fetal Bovine Serum (FBS). Thisaddition of FBS serves to better represent the environment existingin-vivo and predict the ability of OMN peptides to exert their effect inlive animals and later in the human body

FIGS. 6C and 6D: The same experiment was conducted with E. coli NDM-1bacteria, an Imipenem (IPM) resistant strain. IPM concentration is givenin μg/ml. FIG. 6C clearly shows that IPM has lost its inhibitory effecton bacteria growth as a result of the bacteria resistance. In FIG. 6DOMN6 exerts a powerful antimicrobial effect on this resistant strain.These results show that OMN6 is an effective antimicrobial agent againstdrug-resistant bacteria, on which a treatment with conventionalantibiotics has failed.

FIG. 7: is a picture depicting an example of the antimicrobial effect ofOMN6 on resistant bacteria. Multi Drug Resistant A. baumannii bacteriawas plated on appropriate medium and incubated for 24-48 hours to allowthe growth of colonies. Prior to plating, bacteria were incubated withOMN6 at increasing concentrations of between 0-10 μM (see also in FIG.8), and monitored as previously described (FIG. 6 and Example 5).

Bacteria in CTRL, when plated, yielded countless colonies, showing thattheir growth has not been inhibited. When OMN6 treated bacteria wereplated, the medium remained clear and no colonies were seen. The absenceof colonies indicates that all the bacteria were killed as a result ofthe treatment with OMN6.

FIG. 8: is an overview of the preliminary topical safety experiment inmice as detailed in Table 9. All the groups and treatments are presentedas well as all the analysis assays performed. The figure depicts theresults from the analysis of the hemolysis assay conducted. The figuredepicts the quantification of Free-Hemoglobin averaged for eachexperimental group and presented as percent of the control group(%/CTRL).

FIG. 9: Is an overview of the preliminary intraperitoneal (IP) injectionsafety experiment in mice as detailed in Table 10. All the groups andtreatments are presented as well as all the analysis assays performed.This Figure shows the results from the analysis of the hemolysis assayconducted. The figure depicts the quantification of Free-Hemoglobinaveraged for each experimental group and presents it as % of CTRL.

FIGS. 10A-10B summarizes the results of an experiment conducted toevaluate the efficacy of OMN6 in-vivo. Mice were injected subcutaneously(SC) with 10⁸ Colony Forming Units (CFU)/mouse of E. coli ESBL, aresistant strain. Experiment group was treated with OMN6 at aconcentration of 8 mg/kg and CTRL group was treated with saline (0.9%NaCl) solution as a sham treatment.

After four days, mice were sacrificed and skin samples were analyzed forbacterial burden (FIG. 10A) and abscess size in mm² (FIG. 10B). Resultsshow that the bacterial burden was lowered by 94% in the group treatedwith OMN6. Abscess size was decreased by 80% in the group treated byOMN6.

Considering these results, it is clear that OMN6 is exerting a strongantimicrobial effect and is highly efficient in-vivo.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on peptides from the Cecropin family, which areexpressed mainly in insects from the Lepidoptera and Diptera orders.

The present invention provides, degradation-resistant, peptides that canbe used as antibiotic medicaments. In some embodiments, the peptidescomprise at least one cysteine residue at their carboxy- andamino-terminus. In some embodiments of the invention, under oxidativeenvironment, e.g. as in various infections, the cysteines in thecarboxy- and amino-terminus of the peptides of the invention, arecovalently bonded, thus creating a cyclic form of the peptides, whereinsaid cyclic peptides represent higher stability while maintaining itsoriginal biological activity.

In some embodiments, there is provided a peptide comprising: a coreamino acid sequence, which is identical or similar to the amino acidsequence of a member of the Cecropin family, wherein the core amino acidsequence is extended at the N-terminus by an N-terminal group and/orextended at the C-terminus by a C-terminal group; and wherein theN-terminal group and/or the C-terminal group are identical or differentor null and are capable of forming a covalent bond so as to form acyclic peptide or a homomultimer assembly via intermolecular covalentlinkage.

As used herein, in one embodiment the phrase “homomultimer assembly”refers to molecular structural organization comprising more than onereplica of the same molecule. The connectivity and structuralorganization of the different replicas of the same molecule could bemaintained via covalent bond and/or non covalent interactions.

As used herein, in one embodiment the phrase “intermolecular covalentlinkage” refers to covalent bond that is formed between two identical ordifferent molecules.

In some exemplary embodiments of the invention, the member of theCecropin family belongs to the group of AMP CMIV, Cecropin A, CecropinB, Cecropin B2, Cecropin D, Cecropin IA, and Cecropin P1.

Reference is made to Tables 1, 2 and 3 which show a library of 11exemplary antimicrobial peptides of the invention (sequences 1-11, Table1), the original sequences from the Cecropin family (sequences 12-22,Table 2), and nucleic acid sequences encoding the peptides as set forthin SEQ ID Nos. 1-11, Table 3).

Table 1 presents the amino acid sequence of the modified peptides.Inserted cysteine and methionine residues appear in bold.

TABLE 1 Omnix Medical Modified Peptides Species origin/ Original NameSequence ID Cecropin Amino acid sequence OMN 1 SEQ ID NO: 1 S. peregrinaMCGWLKKIGKKIERVGQHTRDA Cecropin IA TIQGLGIAQQAANVAATARGC OMN 2SEQ ID NO: 2 H. cecropia MCKWKVFKKIEMKGRNIRNGIV Cecropin BKAGPAIAVLGEAKALC OMN 3 SEQ ID NO: 3 M. sexta MCWNPFKELERAGQRVRDAVTSCecropin B-2 AAPAVATVGQAAAIARC OMN 4 SEQ ID NO: 4 H. cecropiaMCWNPFKELEKVGQRVRDAVIS Cecropin D AGPAVATVAQATALAKC OMN 5 SEQ ID NO: 5A. pernyi MCWNPFKELERAGQRVRDAIISA Cecropin D GPAVATVAQATALAKC OMN 6SEQ ID NO: 6 H. cecropia MCKWKLFKKIEKVGQNIRDGIIK Cecropin AAGPAVAVVGQATQIAKC OMN 7 SEQ ID NO: 7 B. mori AMP MCRWKIFKKIEKVGQNIRDGIVKCM_(IV) AGPAVAVVGQAATIC OMN 8 SEQ ID NO: 8 B. moriMCRWKIFKKIEKMGRNIRDGIVA Cecropin A AGPAIEVLGSAKAIC OMN 9 SEQ ID NO: 9A. pernyi MCKWKIFKKIEKVGRNIRNGIIK Cecropin B AGPAVAVLGEAKALC OMN 10SEQ ID NO: 10 D. melanogaster MCGWLKKIGKKIERVGQHTRDA Cecropin ATIQGLGIAQQAANVAATARC OMN 11 SEQ ID NO: 11 S.s. domesticusMCSWLSKTAKKLENSAKKRISE Cecropin P1 GIAIAIQGGPRC

Table 2 presents the amino acid sequences of exemplary peptides from theCecropin family and their origin.

TABLE 2 Cecropin Family of Peptides Original Sequence ID Species originCecropin Amino acid sequence SEQ ID NO: 12 S. peregina Cecropin IAGWLKKIGKKIERVGQHTRD ATIQGLGIAQQAANVAATAR G SEQ ID NO: 13 H. cecropiaCecropin B KWKVFKKIEMKGRNIRNGIV KAGPAIAVLGEAKAL SEQ ID NO: 14 M. sextaCecropin B-2 WNPFKELERAGQRVRDAVTS AAPAVATVGQAAAIAR SEQ ID NO: 15H. cecropia Cecropin D WNPFKELEKVGQRVRDAVIS AGPAVATVAQATALAKSEQ ID NO: 16 A. pernyi Cecropin D WNPFKELERAGQRVRDAIIS AGPAVATVAQATALAKSEQ ID NO: 17 H. cecropia Cecropin A KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK SEQ ID NO: 18 B. mori AMP CM_(IV) RWKIFKKIEKVGQNIRDGIVKAGPAVAVVGQAATI SEQ ID NO: 19 B. mori Cecropin A RWKIFKKIEKMGRNIRDGIVAAGPAIEVLGSAKAI SEQ ID NO: 20 A. pernyi Cecropin B KWKIFKKIEKVGRNIRNGIIKAGPAVAVLGEAKAL SEQ ID NO: 21 D. melanogaster Cecropin AGWLKKIGKKIERVGQHTRD ATIQGLGIAQQAANVAATAR SEQ ID NO: 22 S.s. domesticusCecropin P1 SWLSKTAKKLENSAKKRISE GIAIAIQGGPR

Table 3 presents the nucleic acid sequences encoding respectively thepeptides of sequences 1-11.

TABLE 3Nucleotide Sequence encoding the modified peptides of the invention.Species origin/ Original Name Sequence ID Cecropin Nucleotide sequenceOMN 1 SEQ ID NO: 23 S. peregrina atgtgcggctggctgaaaaaaattggc Cecropin IAaaaaaaattgaacgcgtgggccagcat acccgcgatgcgaccattcagggcctgggcattgcgcagcaggcggcgaacgtg gcggcgaccgcgcgcggctgc OMN 2 SEQ ID NO: 24H. cecropia atgtgcaaatggaaagtgtttaaaaaa Cecropin Battgaaaaaatgggccgcaacattcgc aacggcattgtgaaagcgggcccggcgattgcggtgctgggcgaagcgaaagcg ctgggctgc OMN 3 SEQ ID NO: 25 M. sextaatgtgctggaacccgtttaaagaactg Cecropin B-2 gaacgcgcgggccagcgcgtgcgcgatgcggtgattagcgcggcgccggcggtg gcgaccgtgggccaggcggcggcgatt gcgcgcggctgcOMN 4 SEQ ID NO: 26 H. cecropia atgtgctggaacccgtttaaagaactg Cecropin Dgaaaaagtgggccagcgcgtgcgcgat gcggtgattagcgcgggcccggcggtggcgaccgtggcgcaggcgaccgcgctg gcgaaaggcaaatgc OMN 5 SEQ ID NO: 27A. pernyi atgtgctggaacccgtttaaagaactg Cecropin Dgaacgcgcgggccagcgcgtgcgcgat gcgattattagcgcgggcccggcggtggcgaccgtggcgcaggcgaccgcgctg gcgaaatgc OMN 6 SEQ ID NO: 28 H. cecropiaatgtgcaaatggaaactgtttaaaaaa Cecropin A attgaaaaagtgggccagaacattcgcgatggcattattaaagcgggcccggcg gtggcggtggtgggccaggcgacccag attgcgaaaggctgcOMN 7 SEQ ID NO: 29 B. mori atgtgccgctggaaaatttttaaaaaa AMP CM_(IV)attgaaaaagtgggccagaacattcgc gatggcattgtgaaagcgggcccggcggtggcggtggtgggccaggcggcgacc atttgc OMN 8 SEQ ID NO: 30 B. moriatgtgccgctggaaaatttttaaaaaa Cecropin A attgaaaaaatgggccgcaacattcgcgatggcattgtgaaagcgggcccggcg attgaagtgctgggcagcgcgaaagcg attggcaaatgcOMN 9 SEQ ID NO: 31 A. pernyi atgtgcaaatggaaaatttttaaaaaa Cecropin Battgaaaaagtgggccgcaacattcgc aacggcattattaaagcgggcccggcggtggcggtgctgggcgaagcgaaagcg ctgtgc OMN 10 SEQ ID NO: 32 D. melanogasteratgtgcagcgaagcgggctggctgaaa Cecropin A aaaattggcaaaaaaattgaacgcgtgggccagcatacccgcgatgcgaccatt cagggcctgggcattgcgcagcaggcggcgaacgtggcggcgaccgcgcgcggc tgc OMN 11 SEQ ID NO: 33 S.s. domesticusatgtgcagctggctgagcaaaaccgcg Cecropin P1 aaaaaactggaaaacagcgcgaaaaaacgcattagcgaaggcattgcgattgcg attcagggcggcccgcgctgc

In some embodiments of the invention, the cyclic peptide has a topology,wherein the topology is head-to-tail, side-chain-to-side-chain,head-to-side-chain or side-chain-to-tail or backbone-to-backbone orside-chain-to-backbone or head-to-backbone or tail-to-backbone. Thecyclic peptide according to some embodiments of the invention ishomodetic cyclic peptide, cyclic isopeptide, cyclic depsipeptide orbicyclic peptide. In some embodiments, the covalent linkage is formedunder oxidative and/or acidic physiological conditions. In someembodiments, the peptide of the invention is a stapled peptide.

As used herein, in one embodiment relating to the topology of the cyclicpeptide, the phrase “head-to-tail” refer to cyclization of the peptidevia amide bond formation between the amino terminus and the carboxylterminus of the peptide.

As used herein, in one embodiment relating to the topology of the cyclicpeptide, the phrase “side-chain-to-side-chain” refers to cyclization ofthe peptide via the formation of covalent bond between two side chains.

As used herein, in one embodiment relating to the topology of the cyclicpeptide, the phrase “head-to-side-chain” refers to cyclization of thepeptide via the formation of covalent bond between the amino terminusand a side chain of the peptide.

As used herein, in one embodiment relating to the topology of the cyclicpeptide, the phrase “side-chain-to-tail” refers to cyclization of thepeptide via the formation of covalent bond between the carboxyl terminusand a side chain of the peptide.

As used herein, in one embodiment relating to the topology of the cyclicpeptide, the phrase “backbone-to-backbone” refers to cyclization of thepeptide via the formation of covalent bond between two differentbackbone atoms of the peptide.

As used herein, in one embodiment relating to the topology of the cyclicpeptide, the phrase “side-chain-to-backbone” refers to cyclization ofthe peptide via the formation of covalent bond between a side chain anda backbone atom of the peptide.

As used herein, in one embodiment relating to the topology of the cyclicpeptide, the phrase “head-to-backbone” refers to cyclization of thepeptide via the formation of covalent bond between the amino terminusand a backbone atom of the peptide.

As used herein, in one embodiment relating to the topology of the cyclicpeptide, the phrase “tail-to-backbone” refers to cyclization of thepeptide via the formation of covalent bond between the carboxyl terminusand a backbone atom of the peptide.

In some embodiments, the core amino acid sequence of a member of theCecropin family is as set forth in SEQ ID Nos: 12-22 as detailed inTable 2. The core amino acid sequence may comprise of L or Dstereoisomers or combination thereof. In some embodiments of theinvention, the core amino acid sequence has at least 70%, 75%, 80%, 85%,90%, 95% or 99% sequence identity to the amino acid sequences set forthin SEQ ID Nos: 12-22. The core amino acid sequence may compriseaccording to some embodiments, substitution, conservative amino acidsubstitutions, conservatively modified sequence variants, deletion,and/or insertion at one or more position or is in reverse order.

As used herein, in one embodiment the phrase “conservative amino acidsubstitutions” or the phrase “conservatively modified sequence variant”refer to trivial changes in amino acid sequence were one of skill willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified sequence variant”,including where the alteration results in the substitution of an aminoacid with a chemically similar amino acid (conservative amino acidsubstitutions). Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Guidance concerning whichamino acid changes are likely to be phenotypically silent can also befound in Bowie et al., 1990, Science 247: 1306 1310. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles. Typical conservativesubstitutions include but are not limited to: 1) Alanine (A), Glycine(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). Amino acids canbe substituted based upon properties associated with side chains, forexample, amino acids with polar side chains may be substituted, forexample, Serine (S) and Threonine (T); amino acids based on theelectrical charge of a side chains, for example, Arginine (R) andHistidine (H); and amino acids that have hydrophobic side chains, forexample, Valine (V) and Leucine (L). As indicated, changes are typicallyof a minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein.

In some embodiments of the invention, the amino acid sequence of themember of the Cecropin family which forms the core amino acid of thepeptide of the invention comprises 17-144 amino acids. In someembodiments of the invention, the amino acid sequence of the member ofthe Cecropin family comprises 20-140 amino acids. In some embodiments ofthe invention, the amino acid sequence of the member of the Cecropinfamily comprises 25-130 amino acids. In some embodiments of theinvention, the amino acid sequence of the member of the Cecropin familycomprises 20-40 amino acids. In some embodiments of the invention, theamino acid sequence of the member of the Cecropin family comprises 25-30amino acids. In some embodiments of the invention, the amino acidsequence of the member of the Cecropin family comprises 20-50 aminoacids. In some embodiments of the invention, the amino acid sequence ofthe member of the Cecropin family comprises 15-50 amino acids. In someembodiments of the invention, the amino acid sequence of the member ofthe Cecropin family comprises 20-70 amino acids. In some embodiments ofthe invention, the amino acid sequence of the member of the Cecropinfamily comprises 20-100 amino acids. In some embodiments of theinvention, the peptide comprises a C-terminus group and/or an N terminusgroup wherein the C-terminus group and/or the N terminus group comprisesone or more of cysteine, cysteine derivative, an amino acid sequencewhich contains cysteine or a group comprising a thiol moiety or anycombination thereof.

In some embodiments of the invention, the c-terminus group and/or the Nterminus group are each selected from the group consisting of cysteine,cysteine derivative, an amino acid sequence which contains cysteine orany other group comprising a thiol moiety. In some embodiments of theinvention, the N-terminus group comprises the amino acid sequencemethionine-cysteine, methionine-cysteine derivative, methioninederivative-cysteine or methionine derivative-cysteine derivative and theC-terminus group is cysteine or a cysteine derivative. In someembodiments of the invention, the C-terminus group comprises the aminoacid sequence methionine-cysteine, methionine-cysteine derivative,methionine derivative-cysteine or methionine derivative-cysteinederivative and the N-terminus group is cysteine or a cysteinederivative.

In some embodiments of the invention, the N-terminus group and theC-terminus group are covalently bound so as to form a cyclic peptide.The covalent linkage may be a disulfide bond, an amide, lactam orpeptide bond.

In some embodiments of the invention, the c-terminus group and the Nterminus group are each selected from the group of L-amino acids,D-amino acids, non-natural amino acid or amino acid derivative.

In some embodiments of the invention, the peptide is self-assembledwithin a physiological membrane such that the intermolecular covalentlinkage is formed between the N-terminus group of the peptide to theN-terminus or a C-terminus group of an additional identical peptide orwherein the intermolecular covalent linkage is formed between theC-terminus group of the peptide to the N-terminus or a C-terminus groupsof an additional identical peptide.

In some embodiments of the invention, wherein the peptide is as setforth in SEQ ID Nos. 1-11. In some embodiments, the peptide is as setforth in SEQ ID. No. 6, which is also designated here OMN6.

In some embodiments, the peptides of the invention are stabilized by anamide group added to the C-terminus group and or by an acetyl group tothe N-terminus group. In some embodiments, the peptides of the inventionare stabilized by any technique which is known in the art such as theaddition of a non-proteinaceous or proteinaceous moiety.

In an embodiment of the invention, the non-proteinaceous is polyethyleneglycol (PEG) or derivative thereof, polyvinyl pyrrolidone (PVP),albumin, divinyl ether, maleic anhydride copolymer (DIVEMA; andpoly(styrene comaleic anhydride) (SMA), hyaluronic acid (HA), alginicacid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), glyme orpolyisopropylacrylamide or any combination thereof.

In one embodiment, this invention provides a functionally equivalentmolecule that mimics the functional activity of any of the peptide orpeptide variants provided in this invention. The term “functionallyequivalent molecule” refers in the application to any compound such asbut not restricted to peptidomimetic or stapled peptide. Thefunctionally equivalent molecule may be obtained by retro-inverso orD-retro-enantiomer peptide technique, consisting of D-amino acids in thereversed sequence. The functionally equivalent molecule may be obtainedby using amino acid derivative.

As used herein, in one embodiment, the term “amino acid derivative”refers to a group derivable from a naturally or non-naturally occurringamino acid, as described and exemplified herein. Amino acid derivativesare apparent to those of skill in the art and include, but are notlimited to, ester, amino alcohol, amino aldehyde, amino lactone, andN-methyl derivatives of naturally and non-naturally occurring aminoacids. In an embodiment, an amino acid derivative is provided as asubstituent of a compound described herein, wherein the substituent is—NH-G(Sc)—C(0)-Q or —OC(0)G(Sc)-Q, wherein Q is —SR, —NRR or alkoxyl, Ris hydrogen or alkyl, Sc is a side chain of a naturally occurring ornon-naturally occurring amino acid and G is C1-C2 alkyl. In certainembodiments, G is Ci alkyl and Sc is selected from the group consistingof hydrogen, alkyl, heteroalkyl, arylalkyl and heteroarylalkyl.

As used herein, in one embodiment, the term “peptide” may be derivedfrom a natural biological source, synthesized, or produced byrecombinant technology. It may be generated in any manner, including bychemical synthesis. One or more of the amino acids may be modified, forexample, by the addition of a chemical entity such as a carbohydrategroup, a phosphate group, a farnesyl group, an isofamesyt group, a fattyacid group, an acyl group (e.g., acetyl group), a linker forconjugation, functionalization, or other known protecting/blockinggroups.

As used herein, in one embodiment, the term “peptide” may be fragments,derivatives, analogs, or variants of the foregoing peptides, and anycombination thereof. “Fragments of peptides”, as that term or phrase isused herein, include proteolytic fragments, as well as deletionfragments. “Variants of peptides” include fragments and peptides withaltered amino acid sequences due to amino acid substitutions, deletions,or insertions.

Variants may occur naturally or be non-naturally occurring. Examplesinclude fusion proteins, peptides having one or more residues chemicallyderivatized by reaction of a functional side group, and peptides thatcontain one or more naturally occurring amino acid derivatives of thetwenty standard amino acids. These modifications may also include theincorporation of D-amino acids, or other non-encoded amino-acids. In oneembodiment, none of the modifications should substantially interferewith the desired biological activity of the peptide, fragment thereof.In another embodiment, modifications may alter a characteristic of thepeptide, fragment thereof, for instance stability or half-life, withoutinterfering with the desired biological activity of the peptide,fragment thereof. In one embodiment, as used herein the terms “peptide”and “protein” may be used interchangeably having all the same meaningsand qualities.

In one embodiment, peptides of the present invention are purified usinga variety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

In one embodiment, to facilitate recovery, the expressed coding sequencecan be engineered to encode the peptide of the present invention andfused cleavable moiety. In one embodiment, a fusion protein can bedesigned so that the peptide can be readily isolated by affinitychromatography; e.g., by immobilization on a column specific for thecleavable moiety. In one embodiment, a cleavage site is engineeredbetween the peptide and the cleavable moiety and the peptide can bereleased from the chromatographic column by treatment with anappropriate enzyme or agent that specifically cleaves the fusion proteinat this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988);and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

In one embodiment, the peptide of the present invention is retrieved ina substantially pure form.

In one embodiment, the phrase “substantially pure” refers to a puritythat allows for the effective use of the protein in the applicationsdescribed herein.

In one embodiment, the peptide of the present invention can also besynthesized using in vitro expression systems. In one embodiment, invitro synthesis methods are well known in the art and the components ofthe system are commercially available.

In one embodiment, a peptide of this invention is produced

synthetic process. In some embodiments the peptide is produced usingrecombinant DNA technology. A “recombinant” peptide, or protein refersto a peptide, or protein produced by recombinant DNA techniques; i.e.,produced from cells transformed by an exogenous DNA construct encodingthe desired peptide or protein.

In some embodiments, the recombinant peptides, fragments thereof orpeptides are synthesized and purified; their therapeutic efficacy can beassayed either in vivo or in vitro. In one embodiment, the activities ofthe peptides of the present invention can be ascertained using variousassays including inter-alia cell viability, survival of mice, andrecovery of wounds.

In one embodiment, a peptide of this invention comprises at least 20amino acids. In another embodiment, a peptide comprises at least 25amino acids. In other embodiments, a peptide comprises at least 30 aminoacids or at least 50 amino acids or 75 amino acids, or 100 amino acids,or 125 amino acids, or 150 amino acids, or 200 amino acids, or 250 aminoacids or 300 amino acids or 350 amino acids or 400 amino acids.

As used herein, in one embodiment, the terms “peptide” and “fragment”may be used interchangeably having all the same meanings and qualities.As used herein, in one embodiment the term “peptide” includes nativepeptides (either degradation products, synthetically synthesizedpeptides or recombinant peptides) and peptidomimetics (typically,synthetically synthesized peptides), such as peptoids and semipeptoidswhich are peptide analogs, which may have, for example, modificationsrendering the peptides more stable while in a body or more capable ofpenetrating into bacterial cells. Such modifications include, but arenot limited to N terminus modification, C terminus modification, peptidebond modification, including, but not limited to, CH2-NH, CH2-S,CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbonemodifications, and residue modification. Methods for preparingpeptidomimetic compounds are well known in the art and are specified,for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter17.2, F. Choplin Pergamon Press (1992), which is incorporated byreference as if fully set forth herein. Further details in this respectare provided herein under.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(˜CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several locations (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acids such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

As used herein, in one embodiment the term “amino acid” refers tonaturally occurring and synthetic α, β γ or δ amino acids, and includesbut is not limited to, amino acids found in proteins, i.e. glycine,alanine, valine, leucine, isoleucine, methionine, phenylalanine,tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine,glutamine, aspartate, glutamate, lysine, arginine and histidine. Incertain embodiments, the amino acid is in the L-configuration.Alternatively, the amino acid can be a derivative of alanyl, valinyl,leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl,methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl,histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl, β-prolinyl,β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl,β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl,β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl. Asused herein, in one embodiment the phrase “Conservatively modifiedvariants” applies to both amino acid and nucleic acid sequences. “Aminoacid variants” refers to amino acid sequences. With respect toparticular nucleic acid sequences, conservatively modified variantsrefers to those nucleic acids which encode identical or essentiallyidentical amino acid sequences, or where the nucleic acid does notencode an amino acid sequence, to essentially identical or associated(e.g., naturally contiguous) sequences. Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode most proteins. For instance, the codons GCA, GCC, GCG and GCU allencode the amino acid alanine. Thus, at every position where an alanineis specified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations”, which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, silent variations of a nucleic acidwhich encodes a polypeptide is implicit in a described sequence withrespect to the expression product.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant”, including where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Guidance concerning whichamino acid changes are likely to be phenotypically silent can also befound in Bowie et al., 1990, Science 247: 1306 1310. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles. Typical conservativesubstitutions include but are not limited to: 1) Alanine (A), Glycine(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). Amino acids canbe substituted based upon properties associated with side chains, forexample, amino acids with polar side chains may be substituted, forexample, Serine (S) and Threonine (T); amino acids based on theelectrical charge of a side chains, for example, Arginine (R) andHistidine (H); and amino acids that have hydrophobic side chains, forexample, Valine (V) and Leucine (L). As indicated, changes are typicallyof a minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein.

In some embodiments, the peptide of the invention is an isolatedpeptide. As used herein, in one embodiment the term “isolated” meansaltered “by the hand of man” from its natural state; i.e., that, if itoccurs in nature, it has been changed or removed from its originalenvironment, or both.

The peptides of the invention may be assayed for example by an agarosedilution MIC assay, a broth dilution, time-kill assay, or equivalentmethods. Antibiotic activity is measured as inhibition of growth orkilling of a microorganism (e.g., bacteria, fungi).

According to another embodiment of the invention, under oxidativeenvironment, e.g. as in infections, the cysteines in the carboxy- andamino-terminus of the peptide of the invention are covalently attachedto one another, thus creating in some embodiments, a cyclic form of thepeptide, which represents higher stability while maintaining thepeptide's original biological activity or even an improved activitycompared to the original form. In some embodiments, these peptides areused as an antibiotic medicament.

The invention also provides a method for expressing the novel cysteineharboring peptides from the Cecropin family in single cell heterologousexpression systems. The method enables a large scale expression whileavoiding rapid degradation by proteolysis activity.

According to some embodiments of the invention, there is provided anucleic acid sequence encoding the peptide of any one of the sequencesas set for in SEQ ID Nos. 1-11 or a vector comprising the nucleic acidsequence encoding the peptide of any one of the sequences as set for inSEQ ID Nos. 1-11.

A massive production of AMCP's in heterologous expression system such asthe yeast strains Saccharomyces cerevisiae/Pichia pastoris or compatiblebacteria strains, serves in some embodiments of the invention togenerate a 1 class of agents effective against a wide range of bothGram-positive and Gram-negative bacteria which overcome the problem ofantibiotic resistance.

According to one method of the invention, the desired genes, such as,but not limited to, any of the peptides listed in Table 1, e.g. theAMCP6 gene (SEQ ID NO:25), are identified, isolated, and cloned into asuitable vector after the addition of Cysteine codons to each of thecarboxy- and amino-terminus of the genes. The vector is suitable forhigh-quantity expression of the target peptides, e.g. pPIC9K and pET28plasmids, and are transformed into an acceptable target expressionsystem, e.g. BL21, Pichia pastoris, Saccharomyces cerevisiae cells, etc.

Desired genes encoding proteins from the Cecropin family are geneticallyengineered to possess an even number of cysteines located at theircarboxy and amino terminus. Table 1 provides a detailed list of severalpeptides of the Cecropin family, including their naive amino acidsequence and the added Cysteine and Methionine residues.

One method of the invention is the insertion of an ATG codon (encodingthe methionine amino acid) to the 5′ of mature Cecropin genes. Whenunmodified peptides are engineered and cysteine residues are inserteddownstream of native ATG codon.

In another embodiment of the invention, the insertion of desired AMCPgene downstream to a region encoding for 6 histidine residues (His-Tag)is provided. A large number of compatible vectors suitable for thispurpose are known to those of skill in the art. One example of theinvention is the use of the vector pET28a, for the expression of thepeptides in compatible bacterial expression system. The use the 6histidine residues His-Tag is a well-known technique for isolation andpurification of proteins from expression system cells.

The use of eukaryotic expression systems is commonly used for theproduction of foreign proteins. One example of such system is themethanoltrophic Pichia pastoris yeast strain. P. pastoris has beendeveloped into an excellent host system for massive production ofdesired proteins. One of the advantages of using P. pastoris over E.coli bacterial cells is that the proteins of interest are usually foldedcorrectly and secreted to the growth medium. Furthermore, P. pastorisdoes not have the endotoxin problem associated with bacteria especiallywhen concerning antimicrobial peptides as required in this invention.Therefore, one embodiment of the invention is the insertion of AMCPgenes into pPIC9K, a Pichia Vector for multi-copy integration andsecreted expression (Invitrogen). AMCP gene of choice is cloned intosaid expression, resulting in pPIC9K-AMCP (9.5 Kb). Cloning the AMCPgenes downstream to the AOX1 promoter verifies that their expression isunder its regulation. For example, AMCP gene containing ATG-TGC codons,encoding methionine and cysteine respectively, at the 5′ of its naïveorigin; and TGC-codon, encoding cysteine, at its 3′; results in theexpression of desired cyclic peptides of the invention.

According to some embodiment of the invention, there is provided amethod of overcoming inherent or acquired resistance of a microorganismto an antibiotic agent, comprising: contacting the microorganism to thepeptide of the invention as described herein. The microorganism is, insome embodiments, Escherichia coli, Klebsiella Pneumoniaea, Pseudomonasaeruginosa, Salmonella serotype Typhi, Acinetobacter baumannii, a memberof Enterobacteriaceae spp., Pseudomonas spp. Salmonella spp.,Acinetobacter spp. or any combination thereof,

As used herein “inherent resistance” of a microorganism to an antibioticagent refers to a natural resistance to the action of the agent even inthe absence of prior exposure to the agent. (R. C. Moellering Jr.,Principles of Anti-infective Therapy; In: Principles and Practice ofInfectious Diseases, 4.sup.th Edition, Eds.; G. L. Mandell, J. E.Bennett, R. Dolin. Churchill Livingstone, New York USA, 1995, page 200).

As used herein, “acquired resistance” of a microorganism to anantibiotic agent refers to a resistance that is not inhibited by thenormal achievable serum concentrations of a recommended antibiotic agentbased on the recommended dosage. (NCCLS guidelines).

As used herein, “tolerance” of a microorganism to an antibiotic agentrefers to when there is microstatic, rather than microcidal effect ofthe agent. Tolerance is measured by an MBC:MIC ratio greater than orequal to 32. (Textbook of Diagnostic Microbiology, Eds., C. R. Mahon andG. Manuselis, W.B. Saunders Co., Toronto Canada, 1995, page 92).

As noted above, this invention provides methods of treating infectionscaused by a microorganism, methods of killing a microorganism, andmethods of enhancing the activity of an antibiotic agent. In particular,these methods are especially applicable when a microorganism isresistant to an antibiotic agent, by a mechanism, such as tolerance,inherent resistance, or acquired resistance. In this invention,infections are treated by administering a therapeutically effective doseof a cationic peptide alone or in combination with an antibiotic agentto a patient with an infection. Similarly, the combination can becontacted with a microorganism to effect killing.

In some embodiments of the invention, there is provided a method ofdisinfecting a wound comprising contacting the wound with the peptide orthe pharmaceutical composition of the invention. The wound may be insome embodiments, a blister wound, a soft tissue wound, a cutaneousabscess, a surgical wound, a sutured laceration, a contaminatedlaceration, a burn wound, a decubitus ulcer, a stasis ulcer, a legulcer, a foot ulcer, a venous ulcer, a diabetic ulcer, an ischemiculcer, a pressure ulcer, an oral infection, a periodontal disease, apartial thickness burn, or a full thickness burn.

In some embodiments of the invention, there is provided a method oftreating an infection, the method comprising administering the peptideor the pharmaceutical composition of the invention to a subject in needthereof.

In some embodiments of the invention, the invention provides use of thepeptide as described herein or a pharmaceutical composition comprisingthe same in the preparation of a medicament for treating an infection ina subject. The infection may be bacterial, viral- and/or fungalinfection.

As used herein in the specification and in the claims section below, theterm “treat” or “treating” and their derivatives includes substantiallyinhibiting or slowing a pathogen growth, or killing the same. Thepathogen may be selected from bacteria, virus, parasite and pathologicfungi.

According to a method of the invention, the peptide of the inventionshould be used in an effective amount to treat infections in mammals. Asused herein, “effective amount” means an amount necessary to achieve thedesired result. For example, an effective amount of the peptides of theinvention to remove a bacterial infection from a mammal within 3 days, 4days, 5 days, 7 days or 10 days. The “effective amount” for purposesherein is that determined by such considerations as are known in theart. Effective doses may be extrapolated from dose-response curvesderived from in vitro or animal model test systems. It is to beunderstood that the “effective amount” is dependent on the treatedbacteria, the subject's physical condition, etc. Determination ofoptimal ranges of effective amounts of the active ingredient, is withinthe skill of the art.

In some embodiments of the invention, there is provided a pharmaceuticalcomposition comprising the peptide of the invention. The pharmaceuticalcomposition may be in a form of a liquid, cream, gel, paste, powder,emulsion, an ointment, a liniment, a lotion, a transdermal system, aninjection fluid, a suspension, a patch film patch or spray. In someembodiments, the formulation is in a form of capsule or a tablet ordesigned for being injected. The composition may be administered inconjunction with one or more additional anti-inflammatory active agent.

According to an embodiment, the compositions of the present inventionmay be formulated for topical, oral, ocular or pulmonary (e.g. forinhalation) administration. Other formulations are described hereinbelowand are within the scope of the invention.

As used herein a “pharmaceutical composition” refers to a preparation ofthe active ingredients described herein with other chemical componentssuch as physiologically suitable carriers and excipients. The purpose ofthe composition is to facilitate administration of the activeingredients (e.g., the peptides of the invention) to the subject.

As used herein the term “active ingredient” refers to the peptidecompositions accountable for the intended biological effect (i.e., fortreatment or prevention of an infection).

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedto refer to a carrier or a diluent that does not cause significantirritation to the subject and does not abrogate the biological activityand properties of the administered active ingredients. An adjuvant isincluded under these phrases.

Herein, the term “excipient” refers to an inert substance added to thecomposition (pharmaceutical composition or cosmetic composition) tofurther facilitate administration of an active ingredient of the presentinvention.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Techniques for formulation and administration may be found in“Remington: The Science and Practice of Pharmacy” Twentieth Edition,Lippincott Williams & Wilkins, Philadelphia, Pa. (1995). For human oranimal administration, preparations should meet sterility, pyrogenicity,general safety and purity standards comparable to those required by theFDA. Administration of the pharmaceutical formulation can be performedin a variety of ways, as described herein.

Another aspect of the present invention relates to a pharmaceuticalcomposition including a pharmaceutically acceptable carrier and anactive ingredient which is the peptide of the invention. The phrase“active ingredient” refers to any of the peptides, the fragmentsthereof, the functionally equivalent molecule that mimics the functionalactivity of the peptide, or a polynucleotide encoding a peptideaccording to the embodiments of the present invention. Thepharmaceutical composition can contain one or more of theabove-identified active ingredients of the present invention. Typically,the pharmaceutical composition of the present invention will include anactive ingredient of the present invention, as well as apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” refers to any suitable adjuvants, carriers,excipients, or stabilizers, and can be in solid or liquid form such as,tablets, capsules, powders, solutions, suspensions, or emulsions.

Typically, the composition will contain from about 0.01 to 99 percent ofthe active ingredient. In some embodiments, the composition will containfrom about 20 to 75 percent of an active ingredient and will furthercontain adjuvants, carriers and/or excipients. Determination of optimalranges of effective amounts of the active ingredient is within the skillof the art. In some embodiments, the pharmaceutical composition maycomprise about 0.01 to about 100 mg/kg body-weight of the peptide. Insome embodiments, the pharmaceutical composition may comprise about 0.5to about 100 mg/kg body-weight of the peptide. In some embodiments, thepharmaceutical composition may comprise about 100 to about 500 mg/kgbody-weight of the peptide. In some embodiments, the pharmaceuticalcomposition may comprise about 100 to about 300 mg/kg body-weight of thepeptide. Treatment regimen for the administration of the peptide of thepresent invention can also be determined readily by those with ordinaryskill in art. That is, the frequency of administration and size of thedose can be established by routine optimization.

In some embodiments, the pharmaceutical composition is in a form of asolid unit dosage form such as a capsule, tablet and the like, such asan ordinary gelatin type containing the active ingredient thereof of thepresent invention, and a carrier, for example, lubricants and inertfillers such as, lactose, sucrose, or cornstarch. In another embodiment,the active ingredient is tabulated with conventional tablet bases suchas lactose, sucrose, or cornstarch in combination with binders likeacacia, cornstarch, or gelatin, disintegrating agents, such ascornstarch, potato starch, or alginic acid, and a lubricant, likestearic acid or magnesium stearate. The tablets, capsules, and the likecan also contain a binder such as gum tragacanth, acacia, corn starch,or gelatin; excipients such as dicalcium phosphate; a disintegratingagent such as corn starch, potato starch, alginic acid; a lubricant suchas magnesium stearate; and a sweetening agent such as sucrose, lactose,or saccharin. When the dosage unit form is a capsule, it can contain, inaddition to materials of the above type, a liquid carrier such as afatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets can be coatedwith shellac, sugar, or both. A syrup can contain, in addition to activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor.

For oral therapeutic administration, the active ingredient can beincorporated with excipients and used in the form of tablets, capsules,elixirs, suspensions, syrups, and the like.

The active ingredient of the present invention may be orallyadministered, for example, with an inert diluent, or with an assimilableedible carrier, or they can be enclosed in hard or soft shell capsules,or they can be compressed into tablets, or they can be incorporateddirectly with the food of the diet.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and should befluid to the extent that easy syringability exists. It should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (e.g., glycerol, propylene glycol, andliquid polyethylene glycol), suitable mixtures thereof, and vegetableoils.

For use as aerosols, the active ingredient thereof of the presentinvention in solution or suspension may be packaged in a pressurizedaerosol container together with suitable propellants, for example,hydrocarbon propellants like propane, butane, or isobutane withconventional adjuvants. The materials of the present invention also maybe administered in a non-pressurized form such as in a nebulizer oratomizer.

When administering the active ingredient of the present invention, andpharmaceutical compositions thereof, they can be administeredsystemically or, alternatively, they can be administered directly to aspecific site. Thus, administering can be accomplished in any mannereffective for delivering the active ingredients thereof or thepharmaceutical compositions to the specific targeted cells. Exemplarymodes of administration include, without limitation, administering theactive ingredients thereof or compositions orally, topically,transdermally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, by intranasal instillation, byintracavitary or intravesical instillation, intraocularly,intraarterially, intralesionally, or by application to mucous membranes,such as, that of the nose, throat, and bronchial tubes.

Toxicity and therapeutic efficacy of the peptides described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC50 (the concentrationwhich provides 50% inhibition) and the LD50 (lethal dose causing deathin 50% of the tested animals) for a subject compound. The data obtainedfrom these cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, for example, Fingl et al., 1975, in The PharmacologicalBasis of Therapeutics, Ch. 1 p. 1, the contents of which are herebyincorporated by reference in their entirety).

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

The amount of a composition to be administered will, of course, dependon the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, and allother relevant factors. Determination of the exact dose to beadministered is conducted by methods known to a person of skill in theart.

It is further understood that the active ingredient of the invention canbe formulated or administered together with additional activeingredients as required to treat the condition of the patient.

Alternately, one may administer the composition in a local rather thansystemic manner, for example, by injecting the composition including theactive ingredient (and a physiologically acceptable carrier) directlyinto a tissue region of a patient (e.g. to the infected skin or into ahealthy skin that surrounds the infected skin).

Suitable routes of administration of the compositions may, for example,include ocular (e.g., to the eye), topical (e.g., to a keratinoustissue, such as the skin, hair, nail, scalp), transdermal, subdermal,pulmonary and oral (e.g., by mouth) administrations.

According to an embodiment, the composition of the present invention isadministered topically, pulmonary (e.g. via inhalation), orally orocularly.

As used herein the phrase “dermal administration” refers to applying orspreading the composition of the present invention onto the surface ofthe body, i.e. skin, scalp, hair, nails and the like, preferably on thesurface affected by the infection.

As used herein the phrase “transdermal administration” refers toadministration of the compositions of the present invention across theskin for systemic administration (e.g. via transdermal patches or bytransdermal implants). The transdermal administration is typicallyeffected in close proximity to the site of infection, however,transdermal administration may be carried out in any anatomical locationas see fit by one of ordinary skill in the art.

As used herein the phrase “subdermal administration” refers toadministering the compositions of the present invention under the skin(i.e. completely buried in the skin, e.g. via subdermal implants). Thesubdermal administration is typically effected in close proximity to thesite of the infection, however, subdermal administration may be carriedout in any anatomical location as see fit by one of ordinary skill inthe art.

Compositions of the present invention may be manufactured by processeswell known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Compositions for use in accordance with some embodiments of theinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used cosmetically or pharmaceutically. Properformulation is dependent upon the route of administration chosen.

In addition, a dose can be formulated in tissue cultures systems (e.g.ex-vivo systems) or in animal models to achieve a desired concentrationor titer. Such information can be used to more accurately determineuseful doses in humans. For example, a therapeutically effective amountcan be evaluated in-vivo by determining the level of inflammation beforeand after administration of the composition in a subject affected by aninflammatory state [e.g. by use of a blood test such as a complete bloodcount (CBC), by observation of skin wounds and so forth].

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration, and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl, E. et al. (1975), “The PharmacologicalBasis of Therapeutics,” Ch. 1, p. 1.)

Depending on the severity of the condition (e.g., the area, depth anddegree of the infection) and the responsiveness of the subject totreatment, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks,several months or several years, or until cure is effected or diminutionof the infection is achieved. Alternatively, the compositions areadministered in order to prevent occurrence of an infection in a subjectat risk of developing an infection (e.g. a subject suffering from achronic inflammatory disease). The compositions may be administered forprolonged periods of time (e.g. several days, several weeks, severalmonths or several years) as to prevent occurrence of an infection.

According to an embodiment of the present invention, the compositions ofthe present invention are administered at least once a day. According toanother embodiment, the compositions are administered twice a day, threetimes a day or more.

According to an embodiment of the present invention, administering iseffected chronically.

According to another embodiment, administering is effected for at leastabout 10 days, 12 days, 14 days, 16 days, 18 days, 21 days, 24 days, 27days, 30 days, 60 days, 90 days or more.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

The compositions of the present invention may be formulated as a unitdosage form. In such form, the preparation is subdivided into unit dosescontaining appropriate quantities of the active ingredients such as fora single administration. The unit dosage form can be a packagedpreparation, the package containing discrete quantities of preparation,for example, an ampule, a dispender, an adhesive bandage, a non-adhesivebandage, a wipe, a baby wipe, a gauze, a pad and a sanitary pad.

Additional factors may be incorporated into the compositions of thepresent invention (i.e., plant extracts as described hereinabove). Theseinclude, but are not limited to, extracellular matrix components (e.g.vitronectin, laminin, collagen, elastin), growth factors (e.g. FGF 1,FGF 2, IGF 1, IGF 2, PDGF, EGF, KGF, HGF, VEGF, SDF-1, GM-CSF, CSF,G-CSF, TGF alpha, TGF beta, NGF and ECGF), growth factors [e.g.erythropoietin, fibroblast growth factor, franulocyte-colony stimulatingfactor (G-CSF) and granulocyte-macrophage colony stimulating factor(GM-CSF)], hormones (e.g., insulin, growth hormone (GH), CRH, Leptin,Prolactin and TSH), angiogenic factors (e.g., angiogenin andangiopoietin), coagulation and anticoagulation factors [e.g., Factor I,Factor XIII, tissue factor, calcium, vWF, protein C, protein S, proteinZ, fibronectin, antithrombin, heparin, plasminogen, low molecular weightheparin (Clixan), high molecular weight kininogen (HMWK), prekallikrein,plasminogen activator inhibitor-1 (PAI1), plasminogen activatorinhibitor-2 (PAI2), urokinase, thrombomoduline, tissue plasminogenactivator (tPA), alpha 2-antiplasmin and Protein Z-related proteaseinhibitor (ZPI)], cytokine inhibitors (e.g. Cyclosporin A;Alpha-2-Macroglobulin, Pentamidine, Pentoxifylline, Dexamethasone),chemokine inhibitors (e.g. Peptide 3, NR58.3-14-3), enzymes (e.g.endoglycosidases, exoglycosidases, endonucleases, exonucleases,peptidases, lipases, oxidases, decarboxylases, hydrases, chondroitinase,chondroitinase ABC, chondroitinase AC, hyaluronidase, keratanase,heparanases, heparanase splice variance, collagenase, trypsin,catalases), neurotransmitters, neuropeptides (e.g. substance P),vitamins (e.g., D-biotin, Choline Chloride, Folic acid, Myo-inositol,Niacinamide, D-Pantothenic acid, Calcium salts, Pyridoxal.HCl,Pyrodixine.HCl, Riboflavin, Thiamine.HCl, Vitamin B12, vitamin E,vitamin C, vitamin D, vitamin B1-6, vitamin K, vitamin A and vitaminPP), carbohydrates (e.g. Mono/Di/Polysaccharides including glucose,mannose, maltose and fructose), ions, chelators (e.g. Fe chelators, Cachelators), antioxidants (e.g., Vitamin E, Quercetin, superoxidescavengers, Superoxide dismutase, H2O2 scavengers, free radicalsscavengers, Fe scavengers), fatty acids (e.g., Triglycerides,Phospholipids, Cholesterols, free fatty acids and non free fatty acids,fatty alcohol, Linoleic acid, oleic acid and lipoic acid), antibiotics(e.g., Penicillins, Cephalosporins and Tetracyclines), amino acids(e.g., essential and non essential (from A-Z) especially glutamine andarginine), salts (e.g., prurivat salts and sulfate salts), sulfates(e.g. Calcium Sulfate), steroids (e.g., androgens, estrogens,progestagens, glucocorticoids and mineralocorticoids), analgesics,anesthetics, anti-bacterial agents, anti-yeast agents, anti-fungalagents, anti-viral agents, pro-biotic agents, anti-protozoal agents,anti-pruritic agents, anti-dermatitis agents, anti-emetics,anti-inflammatory agents, anti-hyperkeratolyic agents, antiperspirants,anti-seborrheic agents, antihistamine agents, hypoxia inducible factors(e.g. HIF-1 alpha and beta and HIF-2), catecholamines (e.g., Epinephrineand Nor-epinephrine), Nucleosides and Nucleotides (e.g., Purins andPyrimidines), Prostaglandins (e.g. Prostaglandin E2), Leucotriens,Erythropoietins (e.g. Thrombopoietin), Proteoglycans (e.g. Heparansulfate, keratan sulfate), Hydroxyapatites [e.g. Hydroxyapatite(Ca10(PO4)6(OH)2)], Haptoglobins (Hp1-1, Hp2-2 and Hp1-2), Superoxidedismutases (e.g. SOD 1/2/3), Nitric Oxides, Nitric Oxide donors (e.g.nitroprusside, Sigma Aldrich, St. Louis, Mo., USA, Glutathioneperoxidases, Hydrating compounds (e.g. vasopressin), cells (e.g.Platelets), cell medium (e.g. M199, DMEM/F12, RPMI, Iscovs), serum (e.g.human serum, fetal calf serum, fetal bovine serum), buffers (e.g.,HEPES, Sodium Bicarbonate), detergents (e.g., Tween), disinfectants,herbs, fruit extracts, vegetable extracts (e.g. cabbage, cucumber),flower extracts, additional plant extracts, flavinoids (e.g. pomegranatejuice), spices, leafs (e.g. Green tea, Chamomile), Polyphenols (e.g. RedWine), honey, lectins, microparticles, nanoparticles (lyposomes),micelles, calcium carbonate (CaCO3, e.g. precipitated calcium carbonate,ground/pulverized calcium carbonate, albacar, PCC, GCC), calcite,limestone, crushed marble, ground limestone, lime, and chalk (e.g.whiting chalk, champagne chalk, french chalk).

The present formulation may also contain ingredients, substances,elements and materials containing, hydrogen, alkyl groups, aryl groups,halo groups, hydroxy groups, alkoxy groups, alkylamino groups,dialkylamino groups, acyl groups, carboxyl groups, carboamido groups,sulfonamide groups, aminoacyl groups, amide groups, amine groups, nitrogroups, organo selenium compounds, hydrocarbons, and cyclichydrocarbons.

The present formulation may be combined with substances such as benzolperoxide, vasoconstrictors, vasodilators, salicylic acid, retinoic acid,azelaic acid, lactic acid, glycolic acid, pyreuric acid, tannins,benzylidenecamphor and derivatives thereof, alpha hydroxyls,surfactants.

Compositions of some embodiments of the present invention may bebioconjugated to polyethyleneglycol (e.g. PEG, SE-PEG) which preservesthe stability (e.g., against protease activities) and/or solubility(e.g., within a biological fluid such as blood, digestive fluid) of theactive ingredients (i.e. plant extract compositions of the presentinvention) while preserving their biological activity and prolonging itshalf-life.

The carrier utilized in the compositions of the invention can be in awide variety of forms. These include emulsion carriers, including, butnot limited to, oil-in-water, water-in-oil, water-in-oil-in-water, andoil-in-water-in-silicone emulsions, a cream, an ointment, an aqueoussolution, a lotion, a soap, a paste, an emulsion, a gel, a spray or anaerosol.

Methods for preparing compositions having such properties are well knownto those skilled in the art, and are described in detail in Remington'sPharmaceutical Sciences, 1990 (supra); and Pharmaceutical Dosage Formsand Drug Delivery Systems, 6th ed., Williams & Wilkins (1995).

EXAMPLES Example 1

Enhanced Stability of OMN6 in Comparison with Native Cecropin A and BSA.

Proteinase-K (ProtK) was used to assess the stability of OMN6 versusnative Cecropin A (CecA) and Bovine Serum Albumin (BSA) (See FIG. 1 A).10 μg of each protein were incubated with increasing concentrations ofbetween 5-20 ng of ProtK as specified, for 2 hours at 37° C. Sampleswere boiled at 100° C. for five minutes and separated on 15% acrylamidegel. The gel was then stained with Coommassie Blue and excess dye wasremoved over-night. Results clearly show that 20 ng of ProtK wassufficient to completely degrade CecA and BSA (lanes: 3, 9respectively). As can be seen, OMN6 is protected from ProtK proteolysisand was not degraded (lane: 6). Results also show that ProtK at a lowconcentration of 5 ng was sufficient to partially degrade CecA and BSA(lanes: 2, 8 respectively). In lane 2, the CecA band was weaker than theuntreated sample band in lane 1. In lane 8, fragments of degraded BSAwere detected, evidence of partial degradation. OMN6 was not degraded byProtK at all. These results prove that OMN6, after the geneticengineering, is more stable than its native form, Cecropin A.

The same experimental system as described above was used to assess thestability of OMN2, OMN7 and OMN11 against proteolytic degradation byProtK (see FIG. 1B). In each case, as specified, results show that OMNpeptides are not degraded by ProtK. The peptides are stable as can beseen from the equal intensity of the bands presented for each peptide.

Example 2

FIG. 2A: OMN6 Exerts a More Powerful Antimicrobial Effect than NativePeptide Cecropin-A.

An assay was conducted in order to compare the antimicrobial activity ofthe native peptide Cecropin A (CecA) vs. a peptide of the inventionOMN6. E. coli bacteria were cultured with CecA or OMN6 in concentrationof 12.5 μM for 17-20 hours. The growth of the bacteria was continuouslymonitored via spectrophotometry at 600 nm. As bacterial growthprogresses, OD600 nm values rise, and where the growth is inhibitedOD600 nm values remain constant. The results clearly show that at theconcentration of 12.5 μM, the genetically engineered peptide OMN6exerted a strong antimicrobial effect and completely inhibited bacterialgrowth for more than 17 hours, the entire duration of the experiment. Athigher concentrations the bacterial growth was totally inhibited as well(data not shown).

In contrast, when bacteria were incubated under the same experimentalconditions with CecA at 12.5 μM, there was no significant inhibition ofgrowth. The bacteria completely overcame the inhibitory effect of CecAafter 10 hours. The bacteria then continued to thrive and grow todensity similar to that of the CTRL group.

These results taken together serve to strongly suggest that OMN6 is anew and stronger antimicrobial agent than its native counterpart CecA.

FIG. 2B: OMN6 is Stable in Presence of Proteinase-K and Retains itsPotent Antimicrobial Activity.

The susceptibility of OMN6/CecA to proteolytic degradation was assessedand the effects of stability on activity were determined. Nativepeptide-CecA and engineered peptide-OMN6 were incubated with 20 ng ofProteinase-K (ProtK) at 37° C. for two hours (left bars and right bars,respectively). E. coli at 500,000 CFU/ml were incubated for 18 hourswith either CecA or OMN6 pretreated with ProtK. After the incubation,bacterial survival was determined via absorption at OD600 nm and via CFUcount on agar plates.

The results as shown in FIG. 2A show that the genetically engineeredpeptide, OMN6, is a stronger antimicrobial agent than the native formCecA. The inhibition of bacterial growth exerted by CecA is overcomeafter 10 hours while OMN6 inhibits growth for more than 17 hours.Moreover, the effect of OMN6 is of a bactericidal nature as there wereno colonies present when the experiment groups detailed in this examplewere plated. Most importantly, CecA is prone to proteolysis by ProtK andloses its antibacterial activity due to this degradation. It is clearthat CecA treated with ProtK for 2 hours has lost its ability to killbacteria. At 12.5 μM of CecA, bacteria are able to grow to more than 70%of the CTRL-control (non-treated bacteria group) (left bars). When OMN6was treated with 20 ng of ProtK OMN6 was not degraded; rather, wasstable and did not lose its antibacterial activity. At 12.5 μM of OMN6,bacteria growth was inhibited to less than 10% of the CTRL group (rightbars).

Examples 1 and 2 emphasize that OMN6 is a stable peptide. OMN6 is apotent antimicrobial agent even after incubation with a strong proteaselike ProtK. In contrast, the native peptide CecA, which is prone toproteolysis, degrades by proteases and loses its ability to inhibitgrowth or kill bacteria after incubation with ProtK.

Example 3

OMN6 Treatment Leads to Bacteria Cell Lysis and Leakage of GFP fromCells to the Surrounding Media.

In order to determine and evaluate the Mechanism of Action (MOA) bywhich the peptides achieve the remarkable antimicrobial effect theyexert, the following experiments were conducted: GFPuv E. coli bacteriaare a strain that upon induction expresses green fluorescent protein(GFP). The GFP fluorescence can be detected at 395/509 nm, while livebacteria can be detected via absorbance at 600 nm (OD600).

GFPuv E. coli bacteria ubiquitously express GFP in their cytoplasm uponinduction and the fluorescent protein can be detected and visualized.The bacteria were grown and induced to express GFP for three hours, thebacteria were then treated with double distilled water (DDW) or OMN6 andincubated for 30 minutes (FIG. 3A, and FIG. 3B, respectively). At thatpoint, the bacteria were imaged via a microscope (×60 Olympus lens)under UV light. In the CTRL group, treated with DDW, the bacteria wereclearly unharmed. All the bacteria were alive, intact and there was noevidence of GFP leaking from inside the cells to the outside media (FIG.3 A). When the E. coli GFPuv bacteria were treated with 50 μM OMN6 50for 30 minutes, there was massive leaking of GFP from the cells'cytoplasm to the outside media (FIG. 3 B). Since GFP is a protein of 238amino-acid residues (26.9 kDa), this protein is a large protein and itcannot leak from the cells when the plasma membrane is intact. It isevident that OMN6 punctures the membrane and leads to the formation ofpores through which GFP can leak. The formation of these poresconstitutes a physical damage that leads to the bacteria death. Incontrast to small-molecule antibiotics, bacteria do not developresistance against physical disturbance of pore formation in theirplasma membrane.

In another experiment, bacteria were grown and induced to express GFP.The bacteria were treated with DDW (for CTRL) or with OMN6. Bacteriawere then centrifuged (5000 RPM for five minutes) and the pellet wasseparated from the growth media supernatant (see FIG. 4A). Thesupernatant (sup) from the two experiment groups CTRL and OMN6, wasseparated from the pellet and analyzed for the level of fluorescenceunits (FU) (see FIG. 4B). The results clearly show that in the CTRLgroup, where the bacteria remain unharmed, the intact bacterial cellsretain all of the GFP. Fluorescence was detected only inside thebacteria which were concentrated in the pellet at the bottom of thetube. In the group treated with OMN6 50 μM for 30 minutes, the bacteriahave undergone extensive lysis and as a result, GFP leaked out of thecells and was therefore found in the surrounding media. Bothsupernatants were probed for the presence of bacteria via absorbance at600 nm and both supernatants were completely void of bacteria cells(data not shown).

The rationale behind this experiment is that once the sup is free ofbacteria cells, if the peptide causes the lysis of bacterial cells itwill cause the GFP to leak out of the cells into the growth media. Thus,in the CTRL group, where the bacteria are intact, no fluorescence isdetected in the sup while in the sup from the group treated with OMN6,the GFP that leaked out of the lysed cells is detected.

Example 4 OMN2, OMN6, OMN7 and OMN11 do not Present Cytotoxic Effects onHEK293 Cells

Human Embryonic Kidney 293 cells (HEK293) are human originated cellswidely accepted as a model cell-line for evaluating adverse effects ofpotentially hazardous substances. HEK293 cells were cultured to 80%confluency and introduced to increasing concentrations of OMN2, OMN6,OMN7 and OMN11. CTRL group treated with DDW.

After 24 hours, all experiment groups were subjected to Methylene-Blueassay in-order to evaluate and determine cell survival. No significantchanges in cell morphology or survival were observed in all of thegroups (FIG. 5A-D).

Example 5 OMN6 does not Present Cytotoxic Effects on Human PrimaryErythrocytes

Since the peptides of the invention are intended inter-alia to operatein wounds or other damaged tissue, a direct contact with patients' bloodmay occur. Accordingly, an experiment was conducted in order to assesswhether OMN6 has cytotoxic effects on human primary erythrocytes. Thesecells lack any defense mechanism to protect them against damage to theplasma membrane and therefor are a standard model for evaluatingcytotoxic effects on eukaryotic cells. The parameter that was measuredwas the ability of OMN6 to cause hemolysis, red blood cells death, inhuman erythrocytes. The experiments were performed on human bloodsamples using ABX PENTRA DF120 machine routinely used by hospitalpersonnel to conduct such tests. The results show, that hemolysis ofcells did not occur. The erythrocytes did not die as a result of beingcontact with OMN6. The number of cells at the beginning of theexperiment has not been changed throughout the entire duration of theexperiment in any of the experiment groups and the number of cells/mmldid not vary between the CTRL group and the experiment group (Table 4).

The Mean Corpuscular Volume (MCV) of the erythrocytes is a well-acceptedindication of the cells' health and membrane integrity. The experimentwas conducted to assess whether OMN6, which forms pores in bacterialmembranes, has the ability to damage human cells plasma-membrane. As canbe seen from the results demonstrated in Table 5, OMN6 did not cause anydecrease in cell volume throughout the time of the experiment. Theseresults strongly indicate that the peptide does not damage eukaryoticcell-membranes. (Table 5).

OMN6 peptide does not target human membranes of primary erythrocytes andHEK293 cells. No significant reduction in cell counts or other adverseeffect were observed with any of the peptides. These results show thepeptides safety and their highly selective targeting of bacteria cells.

Table 4 and Table 5: hemolysis and Mean Corpuscular Values (MCV) ofhuman primary erythrocytes were evaluated after treatment with OMN6.Treatment with OMN6 did not cause hemolysis of erythrocytes. Cell countwas conducted and the number of cells/ml did not vary between CTRL groupand experiment groups throughout the entire duration of the experiment(Table 4). Erythrocytes Mean Corpuscular Volume (MCV) was determined aswell, MCV values did not change throughout the entire duration of theexperiment and MCV values of experiment group did not vary from those ofCTRL group (Table 5).

TABLE 4 Number of live erythrocytes (cells* 10⁶/ml) Time (min) OMN6 [μM]30 60 120 180 0 1.67 1.68 1.65 1.66 10 1.69 1.66 1.68 1.67 20 1.65 1.701.71 1.70 40 1.60 1.80 1.77 1.76

TABLE 5 Erythrocytes Mean Corpuscular Volume (MCV) Time(min) OMN6 [μM]30 60 120 180 0 65 64 63 64 10 64 64 63 64 20 64 64 63 64 40 64 64 63 64

Example 6

OMN Peptides Minimal Inhibitory Concentration (MIC) Values on Sensitiveand Resistant Bacteria In-Vitro.

In order to determine the MIC values of the peptides, growth andinhibition of various bacteria were monitored after treatment with thepeptides of the invention (Table 6). E. coli bacteria were cultured withor without OMN6 in increasing concentrations of 0.8-200 μM and with orwithout Fetal Bovine Serum 10% for 17-20 hours. The growth of thebacteria was continuously monitored via spectrophotometry at 600 nm. Asbacterial growth progresses, OD600 nm values rise, and where the growthis inhibited OD600 nm values remain constant.

The results clearly show that at Minimal Inhibitory Conc. (MIC) of 12.5μM, the genetically engineered peptide OMN6 exerted a strongantimicrobial effect and inhibited bacterial growth for 17 hours, athigher concentrations the bacterial growth was totally inhibited aswell. When culture media is supplemented with FBS 10%, MIC value of OMN6stands at 6.25 μM (FIG. 6B).

E. coli NDM1 is a Carbapenem resistant strain of bacteria. Theexperimental system described above was used to determine theantimicrobial effect of OMN6 on this bacteria compared to the antibioticdrug Imipenem (IPM), a member of the Carbapenem family of antibiotics.The MIC value for IPM on sensitive E. coli is 4 μg/ml, in a MIC above 8μg/ml the bacteria is considered resistant. FIG. 6C clearly shows thateven when the concentration of IPM is increased to 64 μg/ml, 16 timesthe value of MIC, the growth of the resistant bacteria is not inhibitedat all. Moreover, this strain was also exposed to 128 μg/ml and growthwas not inhibited at all (data not shown). When OMN6 12.5 μM wasintroduced to the system, it completely inhibited bacterial growth (FIG.6D).

These results demonstrate that when bacteria develop resistance to aspecific drug, this drug is no longer effective even at highconcentrations. This drug can no longer be used for therapeutic purposesas it has lost its ability to kill the resistant bacteria.

The bacteria resistance against a specific antibiotic drug or against amultitude of antibiotic drugs does not affect their susceptibility tothe antimicrobial peptides of the invention.

The experimental system and parameters described here were used todetermine the MIC values of OMN2, OMN6, OMN7 and OMN11 on variousbacteria strains (Table 7 and Table 8).

TABLE 6 Bacteria Strains ATCC# Resistance Escherichia coli NDM1 ATCC ®BAA-2452 ™ Carbepenem-resistant (Imipenem and Ertapenem) Escherichiacoli ESBL ATCC ® BAA-198 ™ Multidrug resistant Klabsiella PneumoniaeaNDM1 ATCC ® BAA-2473 ™ Carbepenem-resistant (Imipenem and Ertapenem)Klabsiella Pneumoniaea KPC⁺ ATCC ® BAA-2344 ™ Carbapenem resistant(Imipenem and Ertapenem) Pseudomonas aeruginosa Multidrug resistantATCC ® BAA-2110 ™ Salmonella serotype Typhi Resistant to ampicillinChloramphenicol, ATCC ® 700408 ™ streptomycin, sulfonamide, tetracyclineAcinetobacter baumannii Multidrug resistant ATCC ® BAA-1793

TABLE 7 Summary of MIC values (μM) Bacteria |---------------------+10%FBS------| Strain OMN2 OMN6 OMN7 OMN11 OMN2 OMN6 OMN7 OMN11 E. coliSens. 25922 25 12.5 12.5 25 25 3.3 25 25 E. coli ESBL BAA-198 12.5 5 1010 25 5 12.5 5 E. coli CR BAA-2452 20 12.5 25 20 20 10 40 10 K. pneuKPC⁺ BAA-2344 12.5 5 10 10 50 10 20 10 K. pneu NDM1 BAA-2473 100 25 2550 100 10 40 40 P. aeruginosa BAA-2110 >200 20 50 40 100 40 50 40Salmonella Typhi 700408 200 100 100 100 >200 200 >200 200 A. baumanniiBAA-1793 5 2.5 2.5 5 10 5 10 10

Example 7

OMN Peptides Minimal Bactericidal Concentration (MBC) Values onSensitive and Resistant Bacteria In-Vitro.

In order to determine the Minimal Bactericidal Concentration (MBC)values of the peptides, colony formation of various bacteria strains wasmonitored and determined after treatment with the peptides of theinvention. Multi-Drug Resistant A. baumannii bacteria were cultured withor without OMN6 in increasing concentrations, as is detailed in FIG. 6and Example 6. Immediately afterwards, samples of each experimentalgroup were diluted to 1×10⁴ to 1×10⁶ as necessary, and further, thesamples were plated on appropriate medium-agar plates. All the plateswere incubated at 37° C. for 24-48 hours. Colonies were counted andCFU/ml in the original sample, prior to plating, were calculated anddetermined. An example of the plates, after incubation, is presented inFIG. 7 where the plate on the left side of the image is the CTRL sampletreated with DDW and in the right side of the image increasingconcentrations of OMN6 are presented, as specified therein. The resultsshow abundant growth of bacteria in the CTRL group, no inhibition ofgrowth is detected in the CTRL, as well as in OMN6 at concentrations of0.62 μM and 1.25 μM groups. In the groups treated with OMN6 at 2.5 μM, 5μM and 10 μM the plates are clear, i.e void of colonies.

These results clearly demonstrate the antimicrobial effect of OMN6.Furthermore, the fact that the MIC values (FIG. 6 and Example 6) arevery similar to the MBC values (FIG. 7 and Example 7) points to abactericidal effect of the OMN peptides of the invention. The growth ofthe bacteria was not merely inhibited but the bacteria were killed uponcontact with the peptides.

The experimental system and parameters described here were used todetermine the MBC values of OMN2, OMN6, OMN7 and OMN11 on variousbacteria strains with and without the supplementation of 10% FBS (Table6 and Table 8).

TABLE 8 Summary of MBC values (μM) Bacteria |-----------+10%FBS----------------| Strain OMN2 OMN6 OMN7 OMN11 OMN2 OMN6 OMN7 OMN11 E.coli Sens. 25922 25 12.5 12.5 25 25 3.3 25 25 E. coli ESBL BAA-198 12.55 10 10 25 5 12.5 5 E. coli CR BAA-2452 20 12.5 25 20 20 10 40 10 K.pneu KPC⁺ BAA-2344 12.5 5 10 10 50 10 20 10 K. pneu NDM1 BAA-2473 100 2525 50 100 10 40 40 P. aeruginosa BAA-2110 >200 20 50 40 100 40 50 40Salmonella Typhi 700408 200 100 200 200 >200 200 >200 200 A. baumanniiBAA-1793 5 2.5 2.5 5 10 5 10 10

The combined results from the MIC experiments and the MBC experimentsdetailed above show that OMN peptides of the invention are highlyeffective antimicrobial agents. Treatment with OMN peptides directlyleads to the death of resistant bacteria strains. The fact that MIC andMBC values are identical, points to a strong and rapid bactericidaleffect. This bactericidal effect lowers the occurrence of resistancedevelopment as well as the chance of tolerance.

Example 8

OMN6 Antimicrobial Peptide Preliminary Safety in Mouse Model.

The antimicrobial peptide OMN6 was administered to mice in order toevaluate and quantify whether toxic effects are present. OMN6 peptide(C₁₉₅H₃₃₂N₅₆O₄₉S₃)

was administered topically according to concentrations and groupsdetailed in Table 9.

TABLE 9 OMN6 Preliminary Topical Safety in Mice Experiment OverviewGroup Treatment CTRL OMN6 OMN6 OMN6 OMN6 Topical Sham 0.5 mg/kg 1 mg/kg2 mg/kg 4 mg/kg Administration Food/Water ✓ ✓ ✓ ✓ ✓ consumptionMonitoring Mortality ✓ ✓ ✓ ✓ ✓ Hemolysis analysis ✓ ✓ ✓ ✓ ✓

Mice approved for this experiment: Outbred Hsd: ICR (CD-1®)—weight 18-20g. Saline solution (0.9% NaCl) as sham treatment or OMN6 dissolved insaline were administered directly onto the skin of mice after the hairwas shaved. A total volume of 16 μl was administered to each animal.

In this experiment, the peptide OMN6 of the invention was administeredin a single dose, at four different concentrations (mg/kg). Mortality ofmice in all experiment groups was monitored in comparison to a Sham(CTRL) group. Food and water consumption, post treatment, was monitoredby individual weight measurements once every two days. At the end of theexperiment, blood was taken from all animals and 200 μl of blood wasseparated and plasma was analyzed for erythrocyte hemolysis.

Experimental Map:

-   -   1. Mice (six per group) were given a single-dose administration        on day 1, according to group specification (Table 9). Food and        water consumption (weight analysis) along with mortality of mice        in all groups were monitored for four days post treatment. At        the end of the five day trial, the animals were sacrificed.    -   2. Free Hgb (hemoglobin) assay was performed on day 5, on all        samples from all groups, for erythrocyte hemolysis analysis.    -   3. Water/food availability, temperature and other conditions        remained unchanged between the groups for the entire five day        trial.    -   4. Erythrocyte hemolysis analysis was performed via hemoglobin        assay kit (Sigma-Aldrich, 3 Plaut St. Rehovot, Israel).

The results from the topical administration experiment clearly show thatOMN6 does not exert any toxic or otherwise adverse effects when it isadministered topically. Mortality was not observed in any of the groupsthroughout the entire 5-day experiment. Clinical signs such as:diarrhea, bloody diarrhea, stand-on-end hair, apathy or restlessnesswere not observed in any of the groups throughout the entire 5-dayexperiment. Weight-loss was not observed in any of the groups throughoutthe entire 5-day experiment, and further, in all cages animals gainedweight at a normal rate. No evidence of hemolysis of erythrocytes wasobserved in any of the experiment groups. The results strongly suggestthat the peptides are highly specific and target only bacteria cellswithout harming eukaryotic membranes at all.

IP Administration of OMN6:

OMN6 was administered IP to mice in order to evaluate and quantitate anytoxic effects if present. OMN6 peptide was administered viaintraperitoneal injection according to concentrations and groupsdetailed in Table 10.

TABLE 10 OMN6 Preliminary IP Safety in Mice Experiment Overview GroupTreatment CTRL OMN6 IP Administration Sham 16 mg/kg (4 mM SodiumAcetate) Food/Water consumption ✓ ✓ Monitoring Mortality ✓ ✓ Hemolysisanalysis ✓ ✓

Mice used in this experiment: Outbred Hsd: ICR (CD-1®)—weight 18-20 g.Saline solution (0.9% NaCl) supplemented with 4 mM C₂H₃NaO₂ (sodiumacetate) as sham treatment, or OMN6 dissolved in saline wereadministered as specified in Table 9 A total volume of 100 μl wasadministered to each animal. Mortality of mice in experiment group wasmonitored in comparison to a Sham (CTRL) group. Food and waterconsumption, post treatment, was monitored by individual weightmeasurements once every two days. At the end of the experiment, bloodwas taken from all animals, and a 200 μl volume of complete blood wasseparated and plasma was analyzed for erythrocyte hemolysis.

Experimental Map:

-   -   1. Mice (six per group) were given a single-dose administration        on day 1, according to group specification (Table 10). Food and        water consumption (weight analysis) along with mortality of mice        in all groups were monitored for 3 days post treatment. At the        end of the four day trial, the animals were sacrificed.    -   2. Free Hgb (hemoglobin) assay was performed on day 4, on all        samples from all groups, for erythrocyte hemolysis analysis.    -   3. Water/food availability, temperature and other conditions        remained unchanged between the groups for the entire four day        trial.    -   4. The Erythrocyte hemolysis analysis was performed via        hemoglobin assay kit (Sigma-Aldrich, 3 Plaut St. Rehovot,        Israel).

The results clearly show that OMN6 does not exert any toxic or otherwiseadverse effects when injected IP into mice. Mortality was not observedin any of the groups throughout the entire four-day experiment. Clinicalsigns such as: diarrhea, bloody diarrhea, stand-on-end hair, apathy orrestlessness were not observed in any of the groups throughout theentire four-day experiment. Weight-loss was not observed in any of thegroups throughout the entire four-day experiment, further in all of thecages animals gained weight at a normal rate.

The results demonstrate that the peptides of the invention are safe. Noevidence of hemolysis of erythrocytes was observed in any of theexperiment groups. Histological analysis was conducted (data not shown).No evidence of pathological changes or aberrations was observed in anyof the experiment groups.

Example 9

OMN6 Efficacy in Mouse Model Subcutaneous Infection with E. coli.Animals:

Healthy adult female mice weighing 16-20 g were used. CD-I outbred(Harlan Breeding Labs, Jerusalem, Israel) were used in all experiments.Animals were caged in groups of 5-10 and were maintained on chow(Ralston Purina) and water ad-libitum.

Bacteria:

Escherichia coli (ATCC BAA-198) is an extended spectrum beta-lactamase(ESBL) TEM-26 multi drug resistant strain. Stock cultures were grown inTryptic Soy Broth (TSB) (Becton, Dickinson and company BD. Maryland,USA) at 37°.

Colonies were counted after incubation for 24 hours on TSB-agar (BD),and results were expressed as colony forming units (CFU)/ml.

Inoculum:

All broth cultures were adjusted to 1×10⁸ CFU/animal for the challengedose by adding TSB to a final volume of 50 μl. Bacteria wereadministered via subcutaneous (SC) injection with tuberculin syringescapped with 29-gauge needles. Recipient animals had the right flankshaved and depilated with hair remover cream (ORNA19, Israel). To injectthe inoculum, the needle was inserted SC 1 cm lateral to the thoracicspine and just anterior to the right hind extremity. The needle wastracked anteriorly and SC for 1 cm; then 50 μl of inoculum was injected.OMN6 peptide at 8 mg/kg in a total volume of 80 μl was injected SC afterone hour to the same area. All animals appeared well and fully activewithin 2-4 hours after this procedure. All animals were monitored forfood and water consumption, mortality and any other adverse effects forthe entire 5-day duration of the experiment.

Evaluation of Bacterial Burden:

A 2 cm² area of skin was incised and the inner side, where the abscesswas located, was scraped with a sterile scalpel. Scraped tissue wasfiltered through a 40 μm cell-strainer. Filtered mass was collected,centrifuged at 500 RPM for 2 minutes and a sample from the supernatantwas plated on TBS-agar plates for 24 hours at 37°. Colonies were thencounted and results are presented as CFU/abscess (see FIG. 10A).

Evaluation of Abscesses:

Animals were assessed for the presence and size of abscesses on day 5.The animals were anesthetized (Ketamine 225 mg/kg/Xylazine 6 mg/kg BWIP), the right hind flank area was gently exposed by aseptic technique.Abscesses were recorded and then measured by calipers: the product ofthe longest diameter (D) and corresponding perpendicular diameter (d)was determined as “abscess size” (D×d) findings presented in mm² (seeFIG. 10B).

Results: FIG. 10A:

The bacteria burden found in the group of animals inoculated withbacteria and treated with a sham-saline treatment was: 300 CFU/mouse

The bacteria burden found in the group of animals inoculated withbacteria and treated with OMN6 at 8 mg/kg was 20 CFU/mouse.

The results show 93.3% decrease in bacteria burden, pointing to a strongantimicrobial effect of OMN6 in-vivo. The fact that the measurementswere made after five days, points to a powerful and rapid bactericidaleffect of OMN6 leading to direct death of almost all the bacteria thatwere introduced to the mouse.

To conclude, a single dose of OMN6 was enough to eliminate the bacteria,significantly reducing the bacteria burden and the formation of anabscess. Accordingly, it is clear that OMN6 is effective in-vivo and hasa long lasting effect of a bactericidal nature.

FIG. 10B:

The average size of the abscesses found in the group of animalsinoculated with bacteria and treated with a sham-saline treatment was35.25 mm².

The average size of the abscesses found in the group of animalsinoculated with bacteria and treated with OMN6 at 8 mg/kg was 7.25 mm²

The results show 80% decrease in abscess size, pointing to a strongantimicrobial effect of OMN6 in-vivo. Furthermore, the fact that themeasurements were made after five days points to a powerful and rapidbactericidal effect of OMN6 leading to direct death of all the bacteriathat were introduced to the mouse.

What is claimed is:
 1. A cyclic peptide that has an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence setforth in SEQ ID NO:
 7. 2. The cyclic peptide according to claim 1,wherein the amino acid sequence has at least 99% sequence identity tothe amino acid sequence set forth in SEQ ID NO:
 7. 3. The cyclic peptideof claim 1, wherein the peptide is as set forth in SEQ ID NO:7.
 4. Apharmaceutical composition comprising the cyclic peptide of claim
 1. 5.A method of treating a bacterial infection, the method comprisingadministering the cyclic peptide of claim 1 to a subject in needthereof, wherein the bacterial infection is caused by gram negativebacteria.
 6. The pharmaceutical composition of claim 4, wherein thepharmaceutical composition is in a form of a liquid, cream, gel, paste,powder, emulsion, an ointment, a liniment, a lotion, a transdermalsystem, an injection fluid, a suspension, a film patch or spray.
 7. Thepharmaceutical composition of claim 4, which is in the form of a capsuleor a tablet.
 8. A pharmaceutical composition comprising the cyclicpeptide of claim 1 in conjunction with one or more anti-inflammatoryactive agents.
 9. A method of overcoming inherent or acquired resistanceof a gram negative bacteria to an antibiotic agent, comprising:contacting the gram negative bacteria with the cyclic peptide ofclaim
 1. 10. The method of claim 9, wherein the microorganism is amember of Enterobacteriaceae spp., Pseudomonas spp. Salmonella spp., orAcinetobacter spp., or any combination thereof.
 11. A method ofdisinfecting a cutaneous abscess comprising contacting the cutaneousabscess with the cyclic peptide of claim 1, wherein the infection in thecutaneous abscess is caused by gram negative bacteria.